A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans.

Candida albicans is a common human fungal pathogen that colonizes mucosa and develops biofilm in the oral cavity that causes oral candidiasis. It has been reported that cytochrome P450 enzyme (CYP51), a vital part of the ergosterol synthesis cascade, is associated with Candida infections and its biofilm formation. Thidiazuron, a phenyl-urea cytokinin, exhibits anti-senescence and elicitor activity against fungal infection in plants. However, how Thidiazuron impacts C. albicans biofilm formation is still uncertain. Here, we aimed to investigate the effects of a Thidiazuron against the growth and biofilm formation properties of C. albicans using in silico and in vitro experimental approaches. A preliminary molecular docking study revealed potential interaction between Thidiazuron and amino acid residues of CYP51. Further in vitro antifungal susceptibility test, scanning electron microscopy (SEM) and time kill analysis revealed the anti-fungal activity of Thidiazuron in both dose and time-dependent manner. Crystal violet staining, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay revealed 50% inhibition in C. albicans biofilm by Thidiazuron at concentrations 11 and 19 µM respectively. Acridine orange staining assay visually confirmed the biofilm inhibitory potential of Thidiazuron. The gene expression study showed that Thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis (ERG3, ERG11, ERG25), cell adhesion (ASL3, EAP1), and hyphae development (EFG1, HWP1, SAP5) in C. albicans. Wherease, the expression of negative transcription regulator of hyphae (NRG1) was upregulated (5.7-fold) by Thidiazuron treatment. Collectively, our data suggest that Thidiazuron is a robust antifungal compound and an outstanding biofilm inhibitor, which may promise further therapeutic development due to CYP51 binding and inhibition of ergosterol formation against C. albicans.

Biochemical characterization and essentiality of Plasmodium fumarate hydratase.

Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a well-known reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium Fumarate is generated in large quantities in the parasite as a by-product of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterization of PfFH that may provide leads for the development of specific inhibitors is lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a Ki value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain-dependent.

1.6 kW Yb fiber amplifier using chirped seed amplification for stimulated Brillouin scattering suppression.

In a high power fiber amplifier, a frequency-chirped seed interrupts the coherent interaction between the laser and Stokes waves, raising the threshold for stimulated Brillouin scattering (SBS). Moving the external mirror of a vertical cavity surface-emitting diode laser 0.2 µm in 10 µs can yield a frequency chirp of 5×1017 Hz/s at a nearly constant output power. Opto-electronic feedback loops can linearize the chirp, and stabilize the output power. The linear variation of phase with time allows multiple amplifiers to be coherently combined using a frequency shifter to compensate for static and dynamic path length differences. The seed bandwidth, as seen by the counter-propagating SBS, also increases linearly with fiber length, resulting in a nearly-length-independent SBS threshold. Experimental results at the 1.6 kW level with a 19 m delivery fiber are presented. A numerical simulation is also presented.

Immediate Tube Feeding after Percutaneous Endoscopic Gastrostomy: Early Return to Goal Tube Feeds without Added Complications.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) is frequently performed for delivery of nonoral enteral nutrition (EN) in critically ill patients. Tube-based supplement initiation is often delayed for a variety of reasons despite evidence that EN interruption results in worse outcomes. OBJECTIVE: To determine if early initiation of EN after PEG placement is safe and well-tolerated in critically ill patients and if early initiation of EN results in more goal-accomplished days of EN. DESIGN: A retrospective chart review of patients who underwent PEG and at least 24 hours of EN. Patients were stratified according to time to tube- feed initiation: immediate (< one hour), early (one to four hours), and late (four to 24 hours). RESULTS: 'Ihe three groups were similar with respect to demographics, comorbidities, and 30-day mortality. Sixty-one percent of patients in the immediate group were advanced to the previously-met goal EN rates compared to 24% and 18% in the early and delayed groups, respectively (P < .0001). CONCLUSION: Immediate reinitiation of nonoral EN after PEG procedure is safe and is associated with reaching goal nutrition faster.

Endoscopic Retrograde Cholangiopancreatography in Patients with Previous Pancreaticoduodenectomy: A Single-Center Experience.

BACKGROUND: Performing ERCP in patients with previous pancreaticoduodenectomy (PD) is technically challenging. Balloon-assisted ERCP has recently been recognized as a useful tool in patients with surgically altered anatomies. However, there are few studies that focus on ERCP in post-PD patients. AIM: This study aimed to evaluate the outcome of ERCP in patients in post-PD and the patterns for type of endoscopes used. METHODS: Patients with previous PD who had undergone ERCP were included in this retrospective study. RESULTS: One hundred and thirty ERCP procedures were performed on 47 post-PD patients. The overall success of ERCP was 82.3 % (107/130). Endoscope insertion to the pancreaticobiliary anastomoses was accomplished in 93.8 % (122/130), which resulted in successful completion of ERCP in 87.7 % (107/122) of the procedures: 89.5 % (94/105) in biliary indications and 76.5 % (13/17) in pancreas indications. Using the conventional endoscopes (CEs) led to ERCP success in 66.4 % (71/107) of attempts versus 78.3 % (36/46) with balloon-assisted enteroscopes (BAEs). Among 105 cases in which CEs were initially tried, ERCP was successful in 69 (65.7 %) cases with CEs alone. When CEs failed to reach the pancreaticobiliary anastomoses, the subsequent use of BAEs resulted in a successful ERCP in 16/19 (84.2 %) of attempts. CONCLUSIONS: ERCP in post-PD patients can be performed with a high success rate. We recommend that CEs should be used initially for ERCP in patients with PD and that BAEs be reserved for situation in which CEs have failed to reach the pancreaticobiliary anastomoses.

Cost Savings Associated with the Adoption of a Cloud Computing Data Transfer System for Trauma Patients.

INTRODUCTION: Among transferred trauma patients, challenges with the transfer of radiographic studies include problems loading or viewing the studies at the receiving hospitals, and problems manipulating, reconstructing, or evalu- ating the transferred images. Cloud-based image transfer systems may address some ofthese problems. METHODS: We reviewed the charts of patients trans- ferred during one year surrounding the adoption of a cloud computing data transfer system. We compared the rates of repeat imaging before (precloud) and af- ter (postcloud) the adoption of the cloud-based data transfer system. RESULTS: During the precloud period, 28 out of 100 patients required 90 repeat studies. With the cloud computing transfer system in place, three out of 134 patients required seven repeat films. CONCLUSION: There was a statistically significant decrease in the proportion of patients requiring repeat films (28% to 2.2%, P < .0001). Based on an annualized volume of 200 trauma patient transfers, the cost savings estimated using three methods of cost analysis, is between $30,272 and $192,453.

A Common Symptom With an Uncommon Diagnosis: A Case of Primary Esophageal Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Although it can have gastrointestinal involvement, there are limited recorded cases that show primary esophageal DLBCL. This report discusses the case of an 85-year-old female who initially presented with weight loss associated with dysphagia and was later diagnosed with an esophageal mass by endoscopy. Pathology showed large, atypical lymphocytes, and the final morphologic, immunohistochemical, and molecular findings were most consistent with a diagnosis of primary esophageal DLBCL.

Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases.

Malate (2-hydroxysuccinic acid) and tartrate (2,3-dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and antielimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class-I fumarate hydratase (FH) and L-tartrate dehydratase (L-TTD) are two highly conserved enzymes belonging to the iron-sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only (S)-malate and (S,S)-tartrate as substrates while the structurally similar L-TTD from Escherichia coli accepts only (R)-malate and (R,R)-tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L-TTDs and two-subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L-TTD. The switch in enantiomer preference may be rationalized by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme-substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity.

Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum.

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum.

Diagnostic yield and safety of jumbo biopsy forceps in patients with subepithelial lesions of the upper and lower GI tract.

BACKGROUND: EUS-FNA often fails to make a definitive diagnosis in the evaluation of subepithelial lesions. The addition of jumbo biopsy forceps has the potential to improve diagnostic yield, but published series are limited. OBJECTIVE: To assess the likelihood of definitive diagnosis for subepithelial lesions by using jumbo biopsy forceps during EUS examination. DESIGN: Pooled retrospective analysis. SETTING: 6 tertiary referral centers. PATIENTS: All patients having undergone EUS examination for a subepithelial lesion in which jumbo biopsy forceps were used for tissue acquisition. MAIN OUTCOME MEASUREMENTS: Diagnostic yield of jumbo biopsy forceps use, complication rates, and comparison of diagnostic yield with that of EUS-FNA. RESULTS: A total of 129 patients underwent EUS with jumbo biopsy forceps; 31 patients (24%) had simultaneous EUS-FNA. The lesion locations were stomach (n = 98), esophagus (n = 14), duodenum (n = 11), colon (n = 5), and jejunum (n = 1). The average lesion size was 14.9 mm ± 9.3 mm. Overall, definitive diagnosis was obtained in 87 of 129 patients (67.4%) by using either method. A definitive diagnosis was provided by jumbo biopsy forceps use in 76 of 129 patients (58.9%) and by FNA in 14 of 31 patients (45.1%) (P = .175). The results in third-layer lesions were definitive with jumbo biopsy forceps in 56 of 86 lesions (65.1%) and with FNA in 6 of 16 lesions (37.5%) (P = .047). For fourth-layer lesions, the results with jumbo biopsy forceps were definitive in 10 of 25 (40.0%) and with FNA in 8 of 14 (57.1%) (P = .330). Forty-five of 129 patients (34.9%) experienced significant bleeding after biopsy with jumbo forceps and required some form of endoscopic hemostasis. LIMITATIONS: Retrospective study. CONCLUSIONS: Jumbo forceps are a useful tool for the definitive diagnosis of subepithelial lesions. The greatest benefit appears to be with third-layer (submucosal) lesions. The risk of bleeding is significant.

The Gastrointestinal Stromal Tumor (GIST) of a Pancreatic Cyst.

Gastrointestinal stromal tumors (GISTs) are rare tumors and rarely occur outside of the gastrointestinal (GI) tract. When GISTs originate from outside the GI tract, they are called extra-gastrointestinal stromal tumors (EGISTs). In this article, we discuss a case of a 74-year-old woman who presented due to a growing pancreatic lesion on imaging and was subsequently diagnosed with pancreatic EGIST on endoscopic ultrasound with fine-needle aspiration.

Caecal volvulus in a foramen of Winslow hernia.

Caecal herniation through the foramen of Winslow is an uncommon presentation of internal hernia with an estimated overall incidence of 0.02%. Even rarer still is a caecal volvulus strangulated in the lesser sac, a surgical emergency seldom described in the literature. A woman in her 70s presented with a 1-day history of acute-onset right upper quadrant and epigastric pain associated with nausea and vomiting. Prompt CT imaging revealed caecal volvulus within a foramen of Winslow hernia. The diagnosis was confirmed by laparotomy. A right hemicolectomy was performed and the foraminal defect was closed. We identified eight case reports of this rare entity published within the last 30 years. Our patient was managed in a similar manner and recovered without complication, providing further guidance for the operative management of caecal volvulus in the foramen of Winslow.

Mechanisms of protein evolution.

How do proteins evolve? How do changes in sequence mediate changes in protein structure, and in turn in function? This question has multiple angles, ranging from biochemistry and biophysics to evolutionary biology. This review provides a brief integrated view of some key mechanistic aspects of protein evolution. First, we explain how protein evolution is primarily driven by randomly acquired genetic mutations and selection for function, and how these mutations can even give rise to completely new folds. Then, we also comment on how phenotypic protein variability, including promiscuity, transcriptional and translational errors, may also accelerate this process, possibly via "plasticity-first" mechanisms. Finally, we highlight open questions in the field of protein evolution, with respect to the emergence of more sophisticated protein systems such as protein complexes, pathways, and the emergence of pre-LUCA enzymes.

Uniform binding and negative catalysis at the origin of enzymes.

Enzymes are well known for their catalytic abilities, some even reaching "catalytic perfection" in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate-handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a "substrate-handle-binding-first" mechanism ("binding-first" for brevity) instead of "chemistry-first" and we are, therefore, left to wonder what the role of non-catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back-to-back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as "uniform binding" (parallel stabilization of all enzyme-bound states to the same degree). Indeed, we show that a uniform-binding proto-catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate-handle-binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of "binding-first" enzyme families like the aldolase superfamily.

Creating a Chest Wall Injury and Reconstructive program: A single center experience with rib fractures.

BACKGROUND: New Chest Wall Injury and Reconstructive Centers (CWIRC) are emerging; this study aims to investigate the potential benefits of implementing a CWIRC at a single institution. We hypothesized that patients treated at CWIRC will have improved outcomes. METHODS: We instituted a CWIRC in 2019 at our American College of Surgeons (ACS) Level One Trauma Center. We retrospectively compared trauma patients with rib fractures who presented to our center 18 months before (PRE-C) and 18 months after CWIRC implementation (POST-C). Outcomes measured included mortality, length of stay (LOS), intensive care unit (ICU-LOS), readmission rates, and unplanned ICU admission. RESULTS: There were 192 PRE-C patients, compared to 388 POST-C. The mortality in PRE-C was not significantly different compared to the POST-C group (11.46% vs 8.8%, p=0.308). There were also no differences in LOS, ICU-LOS, readmission, and unplanned ICU admission. ICU utilization was dramatically different: PRE-C 17.8% were admitted to ICU compared to 35.6% POST-C (p<0.0001). CONCLUSIONS: The number of patients admitted with rib fractures to our center nearly doubled after CWIRC establishment. Early diagnosis and triage led to significantly more admissions to higher levels of care. There are trends toward improved outcomes using practice management protocols, albeit with higher ICU utilization. Establishment of a CWIRC should be considered for level 1 ACS trauma centers and as utilization of established CWIRC protocols are increased, patients will have improved outcomes. LEVEL OF EVIDENCE: IV STUDY TYPE: Retrospective chart review.

Retrospective review of the costs of routine pelvic X-rays in a trauma setting.

Multidetector Computed Tomography (MDCT) technology plays an important role in the evaluation of injured patients. At our institution pelvic X-ray (PXR) is obtained routinely on trauma patients. Many also receive MDCT of the abdomen and pelvis for other indications. We hypothesized that there would be a substantial cost savings in adopting a policy of deferring PXR in a hemodynamically normal patient who will also proceed to MDCT for other indications. We retrospectively reviewed the charts of trauma patients from February 1, 2008 to February 1, 2009. We reviewed whether a PXR was done, the result, whether an MDCT was also done, and the presence or absence of pelvic fractures. We collected billing and cost data from various hospital sources. We identified 1,330 patients with PXR between February 1, 2008 and February 1, 2009. Of those patients, 810 (61%) had MDCT after PXR. Sixty-six patients (8.0%) had pelvic fractures; 39 were correctly identified on PXR (59% of fractures). Twenty-seven were detected only by MDCT (41% of fractures); all pelvic fractures were identified on MDCT. Seven hundred and forty-four patients (92% of patients with both PXR and MDCT) had negative PXR and negative MDCT. Using three methods of cost analysis, the estimated cost savings range is from $77,011 to $331,080. MDCT of the pelvis is more sensitive and more specific than PXR. In patients who are hemodynamically normal and asymptomatic, forgoing routine PXR could result in an estimated savings from $77,011 to $331,080, depending on the method used to calculate costs.

Temporary use of a new fully-covered self-expanding metal stent for the management of post-esophagectomy strictures.

Perioperative morbidity rates following esophagectomy for esophageal cancer remain quite high (26-41%) even at high-volume centers. Complications may include stricture at the esophagogastric (EG) anastomosis, as well as tracheo-esophageal or tracheo-gastric fistula formation. Fully-covered self-expanding metal stents (FCSEMS) have only recently been described for use in benign esophageal disease. The use of FCSEMS for the management of postoperative complications following esophagectomy has not been well studied. We report our observations in three consecutive patients that underwent placement and subsequent removal of a new, fully-covered metal stent (Wallflex esophageal stent) for treatment of dysphagia due to a persistent stricture at the EG anastomosis.

Fluoroscopy-guided shaped endobiliary biopsy at endoscopic retrograde cholangiography can accurately diagnose biliary neoplasia: Results from a large cohort.

Background and study aims The sensitivity of using standard endobiliary forceps biopsy to diagnose neoplastic biliary lesions remains low. We have developed a unique biopsy approach, termed fluoroscopy-guided, shaped endobiliary biopsy (FSEB), in which the biopsy forceps are modified to improve diagnostic yield. In this study, we evaluate the diagnostic characteristics of FSEB for endobiliary lesions at endoscopic retrograde cholangiography (ERC). Patients and methods Consecutive patients undergoing FSEB between 1/2001 and 12/2014 were retrospectively enrolled. The identification of neoplastic lesions with FSEB, was the primary endpoint. The gold standard of neoplasia was histopathology, cytology or surgical histopathology. The benign cases were followed up for one year. Results A total of 204 patients undergoing 250 biopsy sessions by FSEB were analyzed. Per-patient analysis was performed and FSEB showed 81.1 % sensitivity and 88.2 % accuracy. FSEB detection of proximal biliary lesions was more sensitive (91.1 % vs 73.2 %, P  < 0.01) and accurate (94.9 % vs 82.2 %, P  < 0.01) compared to distal lesions. No complications from FSEB were reported. Conclusions FSEB shows high accuracy for diagnosis of neoplasia in biliary strictures, especially for proximal lesions. Future prospective randomized controlled studies are merited to further validate the role of FSEB as the first-line sampling tool for evaluation of biliary neoplasm.